1. Technical Field
The field of this invention is physiologically acceptable compositions for use as tissue adhesives and sealants.
2. Background
In many situations, there is a need to bond separated tissues. Sutures and staples are effective and well established wound closure devices. However, there are surgical procedures where classical repair procedures are unsatisfactory, limited to highly trained specialists (e.g. microsurgery), or not applicable due to tissue or organ fragility, inaccessibility (e.g. endoscopy procedures), or fluid loss, including capillary xe2x80x9cweepingxe2x80x9d. Tissue adhesives and sealants have been developed to meet these needs. They may be used to seal or reinforce wounds that have been sutured or stapled, as well as finding independent use. The leading commercial products are fibrin glues and cyanoacrylates. However, both products have significant limitations which have prevented their widespread use.
Cyanoacrylates are mainly used for cutaneous wound closure in facial and reconstructive surgery. The appeal of cyanoacrylates is their speed of bonding, which is almost immediate, and its great bond strength. However, its speed of bonding can be a disadvantage, since glued tissue must be cut again in order to reshape it to the desired conformation. Additionally, it can only be used on dry substrates since its mode of action is through a mechanical interlock, limiting its use as a sealant, and it is relatively inflexible compared to surrounding tissue. Cyanoacrylates are also known to be toxic to some tissues and although it is not considered to be biodegradable, potential degradation products are suspected to be carcinogenic.
Fibrin glues comprising blood-derived fibrinogen, factor-XIII and thrombin function primarily as a sealant and hemostat and have been used in many different surgical procedures within the body. They have been shown to be non-toxic, biocompatible and biodegradable. They are able to control excessive bleeding and decrease fibrosis. However, tissues bonded with fibrin cannot be subjected to even moderate tensile stress without rupturing the bond. It takes about three to ten minutes for an initial bond to develop, but requires about 30 minutes to several hours for full strength to develop. Depending upon the application, the product may also resorb too quickly. Use of recombinantly produced fibrinogen, factor XIII, thrombin and related components (e.g. fibrin, activated factor XIII) has not been demonstrated to improve the setting time or strength of fibrin glues. Fibrin glues derived from heterologous, human and animal, serum may provoke undesirable immune responses, and expose the patient to the potential risk of viral infection. Autologous fibrin glues may be impractical to obtain and use and may compromise patient safety.
There is, therefore, substantial interest in developing products which have the biocompatibility of fibrin glues, but which set more quickly and have enhanced strength. These products should be readily available, desirably from other than natural sources, be easily administered and capable of resorption over time.
Relevant Literature
Tissue adhesives are described in: Tissue Adhesives in Surgery, Matsumoto, T., Medical Examination Publishing Co., Inc. 1972 and Sierra, D. H., J. Biomat. App. 7:309-352, 1993. Methods of preparation of protein polymers having blocks of repetitive units are described in U.S. Pat. No. 5,243,038 and EPA 89.913054.3.
Polymeric compositions and methods for their use are provided, where the polymeric compositions are capable of in situ chemical crosslinking to provide novel crosslinked polymeric products, which have good mechanical and biological properties, as exemplified by strong adherent bonds to tissue. The compositions can be used in a variety of applications related to their physical, chemical and biological properties, to bond to separated tissue to provide at least one of the characteristics of a stable, flexible, resorbable bond.
The subject compositions comprise high molecular weight recombinant polymers having one or a combination of repeating units related to naturally occurring structural proteins. Of particular interest are the repeating units of fibroin, elastin, collagen, and keratin, particularly collagen and combinations of fibroin and elastin. The polymers have functional groups which can be chemically crosslinked under physiological conditions with physiologically acceptable crosslinkers, so as to form a composition which has strong adherent properties to a variety of substrates, has strong mechanical properties in maintaining the joint between the substrates, and can be formulated to have good resorption properties.
Of particular interest, the subject compositions provide strongly adherent bonds to tissue to maintain separated tissue in a contiguous spacial relationship. The subject compositions may also be employed as sealants, where the compositions may serve to fill a defect void in tissue, to augment tissue mass or bond synthetic materials to tissues. The subject compositions may also serve as depots in vivo by being mixed with a drug composition, either when used as an adhesive for bonding tissue together or for other bonding or solely as a slow release source of the drug.
The functionalities for crosslinking may be all the same or combinations of functionalities and may include the functionalities of naturally occurring amino acids, such as amino, e.g. lysine, carboxyl, e.g. aspartate and glutamate, guanidine, e.g. arginine, hydroxyl, e.g. serine and threonine, and thiol, e.g. cysteine. Preferably, the functionality is amino (including guanidine).
The polymers will have molecular weights of at least about 15 kD, generally at least about 30 kD, preferably at least about 50 kD and usually not more than 250 kD, more usually not more than about 150 kD. The polymers will have at least two functionalities, more usually at least about four functionalities, generally having an equivalent weight per functionality in the range of about 1 kD to 40 kD, more usually in the range of about 3 kD to 20 kD, preferably in the range of about 3kD to 10 kD, there being at least 3, usually at least 6, functionalities available for crosslinking. If desired, one may use mixtures of polymers, where the polymers have combinations of functionalities or have different functionalities present e.g. carboxyl and amino, thiol and aldehyde, hydroxyl and amino, etc. Thus, depending upon the functionalities and the crosslinking agent, one can form amides, imines, ureas, esters, ethers, urethanes, thioethers, disulfides, and the like.
The individual units in the polymer may be selected from fibroin, GAGAGS (SEQ ID NO:01); elastin, GVGVP (SEQ ID NO:02); collagen GXX, where the X""s may be the same or different, and at least 10 number % and not more than 60 number % of the X""s are proline, and keratin, AKLK/ELAE (SEQ ID NO:3). The desired functionality may be substituted for one of the amino acids of an individual unit or be present as an individual amino acid or part of an intervening group of not more than about 30 amino acids, usually not more than about 16 amino acids. In the former case, within a block of repeats, one or more of the repeats is modified to introduce a crosslinking functionality which would otherwise not normally be present. Thus a valine may be replaced with a lysine, a glycine with an arginine, an alanine with a serine, and the like. In the latter case, there would be an intervening functionality between a block of repeat units, where the number of intervening functionalities would be based on the ranges indicated previously.
Of particular interest are copolymers, either block or random, preferably block, where in the case of elastin and fibroin, the ratio of elastin units to fibroin units is in the range of 16-1:1, preferably 8-1:1, where blocks may have different ratios. Normally, in block copolymers, each block will have at least two units and not more than about 32 units, usually not more than about 24 units. By substituting an amino acid in the unit with an amino acid having the appropriate functionality, one can provide for the appropriate number of functionalities present in the polymer or employ intervening groups between blocks.
The individual amino acid repeat units will have from about 3 to 30 amino acids, usually 3 to 25 amino acids, more usually 3 to 15 amino acids, particularly 3 to 12 amino acids, more particularly about 3 to 9 amino acids. At least 40 weight %, usually at least 50 weight %, more usually at least 70 weight %, of the protein polymer will be composed of segments of repetitive units containing at least 2 identical contiguous repetitive units. Generally repeat blocks will comprise at least 2, 4, 7 or 8 units, and combinations thereof, where copolymers are employed, where the unit which is modified with the crosslinking functionality is counted as a unit.
While for the most part, the polymers of the subject invention will have the active functionality of a naturally occurring amino acid in the chain of the polymer, if desired, pendent groups may be employed to provide the desired functionalities. For example, carboxyl groups may be reacted with polyamines so as to exchange a carboxyl functionality for a single amino or plurality of amino groups. An amino group may be substituted with a polycarboxylic acid, so that the amino group will be replaced with a plurality of carboxylic groups. A thiol may be replaced with an aldehyde, by reaction with an aldehydic olefin, e.g. acrolein, so as to provide for an aldehyde functionality. Other functionalities which may be introduced, if desired, include phosphate esters, activated olefins, e.g. maleimido, thioisocyanato, and the like. The functionalities may be greatly varied from those which naturally occur to provide opportunities for crosslinking. In some instances, this may be desirable to increase the number of functionalities per unit molecular weight, while not increasing the number of functionalities along the chain, for replacing one functionality with another, e.g. thiol with aldehyde, allowing for greater variation in the choice of crosslinking agent.
The crosslinking agent will normally be difunctional, where the functionalities may be the same or different, although higher functionality may be present, usually not exceeding four functionalities. Depending upon the particular functionalities available on the polymers, various crosslinking agents may be employed. The crosslinking agents will usually be at least about three carbon atoms and not more than about 50 carbon atoms, generally ranging from about 3 to 30 carbon atoms, more usually from about 3 to 16 carbon atoms. The chain joining the two functionalities will be at least one atom and not more than about 100 atoms, usually not more than about 60 atoms, preferably not more than about 40 atoms, particularly not more than about 20 atoms, where the atoms may be carbon, oxygen, nitrogen; sulfur, phosphorous, or the like. The linking group may be aliphatically saturated or unsaturated, preferably aliphatic, and may include such functionalities as oxy, ester, amide, thioether, amino, and phosphorous ester. The crosslinking group may be hydrophobic or hydrophilic.
Various reactive functionalities may be employed, such as aldehyde, isocyanate, mixed carboxylic acid anhydride, e.g. ethoxycarbonyl anhydride, activated olefin, activated halo, amino, and the like. By appropriate choice of the functionalities on the protein polymer, and the crosslinking agent, rate of reaction and degree of crosslinking can be controlled.
Various crosslinking agents may be employed, particularly those which have been used previously and have been found to be physiologically acceptable. Crosslinking agents which may be used include dialdehydes, such as glutaraldehyde, activated diolefins, diisocyanates such as, tetramethylene diisocyanate, hexamethylene diisocyanate, octamethylene diisocyanate, acid anhydrides, such as succinic acid dianhydride, ethylene diamine tetraacetic acid dianhydride, diamines, such as hexamethylene diamine, cyclo(L-lysyl-L-lysine), etc. The crosslinking agent may also contain unsymmetrical functionalities, for example, activated olefin aldehydes, e.g. acrolein and quinoid aldehydes, activated halocarboxylic acid anhydride, and the like. The crosslinking agents will usually be commercially available or may be readily synthesized in accordance with conventional ways, either prior to application of the adhesive or by synthesis in situ.
In some instances it may be desirable to react a physiologically acceptable second compound, which serves as a modifying unit, with a polyfunctional, usually bifunctional, compound to change the nature of the crosslinking. The addition of the second compound may be to enhance the rate of crosslinking, change the solubility properties of the crosslinker, enhance or reduce the strength of the crosslinked polymer, enhance or reduce the resorption rate, or provide for other physical, chemical or biological properties of interest. The polyfunctional second compound may be reacted with the crosslinking compound prior to reaction with the protein or concurrently with the reaction with the protein. Where the reaction is prior, the resulting crosslinking product will be physiologically acceptable and when concurrent, the polyfunctional second compound, the crosslinking compound and the resulting crosslinking product will be physiologically acceptable, when used in vivo. The ratio of the polyfunctional second compound to the crosslinking compound will generally be in the range of about 0.1-2:1, more usually in the range of about 0.1-1:1, depending on the reactivity of the polyfunctional second compound when the polyfunctional second compound and crosslinking agent are brought together, the number of crosslinks desired in the final protein composition, the size of the bridge between protein molecules, and the like.
The nature of the polyfunctional second compound may vary widely. The functional groups present may be the same or different from the functional groups present on the polymer, but will be reactive with the functionalities of the crosslinking compound. For example, the polyfunctional second compound may have amino and/or hydroxyl groups, where the protein has amino or hydroxyl functionalities. By employing a diisocyanate with a diol, diurethanes will be produced. Thus, the chain crosslinking the proteins will comprise 2 or more urethanes.
In many instances, the polyfunctional second compound will include an internal functionality that does not participate in the reaction, but provides various other characteristics to the crosslinking agent or the crosslinked protein product. Characteristics of interest include hydrophilicity, hydrolytic instability, sensitivity to enzymatic degradation, biocompatibility, shear strength, and the like. For the most part internal functionalities will comprise oxygen, sulfur and nitrogen atoms, such as ethers, carboxylic acid esters, including urethanes, amino groups, amides, ketones, dithiols and the like. To enhance the rate of resorption, ester groups are of interest, while to enhance hydrophilicity, the same groups maybe employed as well as ethers, such as polyoxyalkylene groups.
The polyfunctional second compound will generally have at least 2 carbon atoms and not more than 50 carbon atoms, usually not more than about 30 carbon atoms, desirably having not more than about 16 carbon atoms per heteroatom. Naturally occurring or synthetic bifunctional compounds may be employed. Illustrative compounds include lysine, arginine, di-(2xe2x80x2-aminoethyl) malonate, citrate, lysyl lysine, 2xe2x80x2-aminoethyl glycinate, O, N-diglycinyl ethanolamine, diethylene glycol diglycinate, cystine, and the like. To provide terminal amino groups, various low molecular weight amino acids may be used, particularly glycine and alanine bonded to an intervening difunctional compound, such as ethylene glycol, diethylene glycol, and tetraethylene glycol, propanediol, 1,4-butyn-2-diol, ascorbic acid, etc.
The subject compositions may be prepared prior to the use of the adhesive by combining the protein polymer and the crosslinking agent, where one or both may have extenders. The two compositions may be readily mixed in accordance with conventional ways, for example, using syringes which can inject the ingredients into a central reactor and the mixture mixed by drawing the mixture back into the syringes and moving the mixture back and forth. Alternatively, the two compositions may be dispensed simultaneously at the site of application. In some instances it may be desirable to allow the crosslinking agent to partially react with the protein prior to adding the polyfunctional second compound. Alternatively, one may mix the polyfunctional second compound with the protein prior to mixing with the crosslinking agent.
Usually, the polymer will be available as a dispersion or solution, particularly aqueous, generally the concentration of the protein polymer being in the range of about 50 mg to 1 g/ml, more usually from about 100 to 800 mg/ml. The solution may be buffered at a pH which enhances or retards the rate of crosslinking. Usually the pH will be in the range of about 2 to 12, more usually 8 to 11. Various buffers may be used, such as phosphate, borate, carbonate, etc. The cation can have an effect on the nature of the product, and to that extent, the alkali metals potassium and sodium, are preferred. The protein composition will generally be about 5 to 40, more usually from about 5 to 20, preferably from about 10 to 20 weight %, to provide for a composition which may be readily handled, will set up within the desired time limit, and the like. The buffer concentration will generally be in the range of about 50 to 500 mM. Other agents may be present in the protein solution, such as stabilizers, surfactants, and the like. If the polyfunctional second compound is present its concentration will be determined in accordance with its ratio to the crosslinking agent and the polymer.
The ratio of crosslinking agent to polymer will vary widely, depending upon the crosslinking agent, the number of functionalities present on the polymer, the desired rate of curing, and the like. Generally, the weight ratio of polymer to crosslinking agent will be at least about 1:1 and not greater than about 100:1, usually not greater than about 50:1, generally being in the range of about 2 to 50:1, but in some instances may not be more than 30:1. The equivalent ratio of protein to crosslinking agent will generally be in the range of about 0.1-1:3, more usually in the range of about 0.5-2:2. Considerations in selecting the protein-crosslinking agent equivalent ratio will be the rate of setup, reactivity of the crosslinking agent, relative solubility of the crosslinking agent in the mixture, physiological properties of the crosslinking agent, desired degree of stability of the crosslinked product, and the like.
If desired, various extenders or extending agents may be used, particularly naturally occurring proteins. Such extenders will usually not exceed 50 weight percent of the composition, generally not exceeding about 20 weight percent, more usually not exceeding about 10 weight percent. Extenders which may be employed include, but are not limited to: synthetic polymers, both addition and condensation polymers, both protein and non-protein, such as polylactides, polyglycolides, polyanhydrides, polyorthoesters, polyvinyl compounds, polyolefins, polyacrylates, polyethylene glycol, polyesters, polyvinyl alcohol, polyethers, copolymers and derivatives thereof; and naturally occurring polymers, such as proteins and non-proteins, including collagen, fibrinogen, fibronectin, laminin, keratin, chitosan, heparin, dextran, alginates, cellulose, glycosoaminoglycans, hyaluronic acid, polysaccharides, derivatives thereof, and the like. The extenders may modulate the setting time and provide for desirable physical or physiological properties of the adhesive.
Based on the lap shear tensile strength test described in the experimental section, within 30 minutes, usually within 15 minutes, more usually within 5 minutes, the lap shear tensile strength will be at least 100, preferably at least about 250, more preferably at least about 300, usually not exceeding about 4000, more usually not exceeding about 3000 g/cm2.
The subject compositions may be applied to the tissue in any convenient way, for example by using a syringe, catheter, cannula, manually applying the composition, spraying or the like. The subject compositions may be applied to the tissue prior to or during the time the tissue segments are held in contiguous relationship. The subject compositions will rapidly develop substantial shear strength, so as to maintain the tissue in proximity. In some situations there will be an interest in having the composition decompose after some reasonable period of time, usually at least one week and generally not more than about four weeks.
Tissues of interest include vascular vessels such as an artery, vein or capillary, muscel, nerve, organs, e.g. liver, spleen, etc., lung, dura, colon, and the like.
In addition to their use as adhesives, the subject compositions may be used to seal or fill defects, e.g. voids or holes, in tissue, and therefore find use as sealants. Thus, the compositions may serve to stop or staunch the flow of fluid, e.g. blood, through ruptured vessels, e.g. arteries, veins, capillaries and the like. In using the subject compositions as sealants, the composition will be applied, as described above, at the site of the defect, whereby it will set and seal the defect. The compositions may be injected into normal or abnormal tissues to augment the tissue mass, e.g. dermis.
The subject compositions may also find use in the formation of articles of manufacture, by themselves or in combination with other materials. In one application, articles may be produced for use internally to a mammalian host, where there is an interest in biocompatibility, reabsorption rate, ability to vascularize, tissue adhesive and/or bonding capability, and the like. Various articles can be prepared, such as gels, films, threads, coatings, formed objects such as pins and screws, or injectable compositions which are flowable, where the injectable composition may set up and bond or seal tissues, form a depot for a drug, augment tissue or be a filler, coating or the like. The formed objects may be prepared in accordance with conventional ways, such as molding, extrusion, precipitation from a solvent, solvent evaporation, and the like. The flowable depot can be obtained by using a molecular dispersion, fine particles in a medium saturated with a polymer, using a melt, where the melting temperature may be achieved by adding physiologically acceptable additives, and the like.
The articles may find use in a variety of situations associated with the implantation of the article into a mammalian host or the application of the article to the surface of a mammalian host, e.g. wound healing, burn dressing, etc. Those situations, where the performance of the articles may be retained for a predetermined time and replaced by natural materials through natural processes, desirably employ materials which will be resorbed after having fulfilled their function in maintaining their role until the natural process has reestablished a natural structure. Thus, the compositions may find use in holding tissue together, covering tissue, encapsulating cells for organs, providing a coating that cells can invade and replace the composition with natural composition, e.g., bone, soft tissues and the like.
To enhance the rate of curing of the polymeric composition, the composition may be partially prepolymerized. When prepolymerized, the polymer will usually have at least about 3% of the total number of crosslinks and not more than about 75% of the total number of crosslinks, as compared to completion of the crosslinking action. The number of crosslinks should allow the resulting product to be workable and provide sufficient time prior to set up for it to be manipulated and used. Alternatively, one may react the functional groups with an excess of the crosslinking reagent, so that the effect is to substitute the functionality of the protein with the functionality of the crosslinking agent. The protein with the substituted functionality may then be used to crosslink protein with the original functionality or with a polyfunctional second compound.
The subject compositions may also be used as depots to provide for a relatively uniform release of a physiologically active product, e.g., a drug. The drug may be mixed with a subject composition at an appropriate concentration prior to crosslinking. As the crosslinked polymer is degraded, the drug will be released due to diffusion as well as erosion of the external surface of the depot. By controlling the form or shape of the depot, the degree of crosslinking, the concentration of the drug and the like, a physiologically therapeutic level of the drug may be maintained over extended periods of time. The period required for absorption can be as short as 0.5 day and may exceed 4, 6 or 8 weeks or more, depending upon the particular composition and the application.
The protein polymer compositions may be prepared in accordance with conventional ways. See, for example, U.S. Pat. No. 5,243,038, which disclosure is incorporated herein by reference. Briefly, sequences may be synthesized comprising a plurality of repeating units, where complementary sequences result in dsDNA having overhangs. A series of dsDNA molecules may be prepared and stepwise introduced into a cloning vector as the gene for the protein is constructed. A monomer can be obtained in this way, which may be sequenced to ensure that there have been no changes in the sequence, followed by multimerization of the monomer, cloning and expression. For further details, see the above indicated patent.
The following examples are offered by way of illustration and not by way of limitation.